The present invention is directed to immunoassay methods and apparatus, and more particularly concerns an immunobead-flow cytometry method, apparatus, assay, device, system, kit, and the like for detecting and quantifying antigens or antibodies and especially adapted for the detection of autoantibodies to nuclear antigens associated with autoimmune diseases.
Typically, autoimmune testing for Systemic Lupus Erythematosis (SLE), Systemic Rheumatic Disease, rheumatoid arthritis, Sjogren's Syndrome, Progressive Systemic Sclerosis (PSS), Subacute Erythematosis, congenital complete heart block, neonatal complete heart block, neonatal lupus dermatitis, Polymyositis, Human Immunodeficiency Virus (HIV), Acquired Immunodeficiency Syndrome (AIDS), as well as other diseases has involved the use of extractable nuclear antigens (ENA) and immunological assays including hemagglutination, counter immunoelectrophoresis (CIE), immunodiffusion, Enzyme Linked Immunosorbent Assay (ELISA), and the like. For example, the Ro(SS-A) antigen having one major band at 60 kD by SDS gel electrophoresis (silver stain) has been purified through the use of immobilized human anti-Ro(SS-A) immunoglobulins. La(SS-B) antigen has two major bands, one at 40 kD and the other at 23 kD (a degradation product) by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-La(SS-B) immunoglobulin. Smith (Sm) antigen has two major bands in the 10 and 14 kD region by SDS gel electrophoresis (silver stain) has been purified through the use of immobilized human anti-Sm (Smith) immunoglobulins. Smith (Sm/RNP) antigen has five bands, one each at 70, 40, 24, 12 and 10 kD, respectively, by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-RNP immunoglobulin. Scl-70 antigen has one major band at 68 kD by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-Scl-70 immunoglobulins. Jo-1 antigen has one major band at 50 kD by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-Jo-1 immunoglobulins. dsDNA double-stranded (native) deoxyribonucleic acid, ssDNA single-stranded DNA, whole Histones, Histone subclasses (distinct molecular fractions) tissue extracts, human antibodies, animal tissue acetone powders, sera and immunoglobulin fractions, second antibodies, anti-whole sera, whole antisera to animal proteins and to human proteins have been used in enzyme immunoassay (ELISA) for detecting or evaluating systemic rheumatic diseases. Immunovision, Inc. of Springdale, Ark. has developed a number of enzyme-linked immunoassays, ouchterlony immunoprecipitation assays, and Western blot assays for detecting human antibody to particular nuclear antigens (ENA).
The presence of human autoantibodies to nuclear antigens, for example, antibodies against RNP/Sm, Sm, SS-A, SS-B, and Scl-70 antigens have been diagnostic when evaluating patients with Systemic Lupus Erythematosis (SLE). Positivity may indicate more progressive disease states or simply rheumatoid arthritis. Currently, enzyme linked immunosorbent assay (ELISA) has been the assay of choice to detect these antibodies. Antibodies to Smith (Sm) antigen have been shown to occur in twenty-five to thirty percent of patients with Systemic Lupus Erythematosis. Antibodies to Sm are less commonly found in patients with other rheumatic diseases. Antibodies to ribosomal nuclear protein (nRNP) have been found in patients with Systemic Lupus Erythematosis. They are also found in sera from patients with rheumatoid arthritis, Sjogren's Syndrome (SS), Progressive Systemic Sclerosis (PSS), and Mixed Connective Tissue Disease (MCTD). Twenty to thirty percent of the patients with antibodies to Scl-70 antigen have Progressive Systemic Sclerosis. Antibodies to Scl-70 are rarely found in patients with other systemic rheumatic diseases. Antibodies to Ro (SS-A) antigen are found in half of Systemic Lupus Erythematosis patients, most patients with Sjogren's Syndrome or Subacute Lupus Erythematosis and nearly all mothers of infants with congenital complete heart block or Neonatal Lupus Dermatitis. Antibodies to the La (SS-B) antigen usually occur in twenty to thirty percent of Sjogren's Syndrome patients and with five to ten percent of Systemic Lupus Erythamatosis patients. Antibodies to Jo-1 antigen are usually found in patients with polymyositis. Antibodies to Ribosomal P antigens are found to occur in five to ten percent of Systemic Lupus Erythematosis patients and ninety percent of those patients will demonstrate signs of lupus psychosis. Antibodies to mitochondrial antigens are found in all primary biliary cirrhosis patients. Antibodies to histone antigens (H1, H2A, H2B, H3, H4) are found in ninety-five to one-hundred percent of drug-induced Lupus Erythematosis, fifteen to twenty percent rheumatoid arthritis, and thirty percent of all patients with Systemic Lupus Erythematosis. Antibodies to cytoplasmic components of neutrophil granulocytes are present in the serum of patients with acute Wegener's granulomatosis and microscopic polyarteritis. Myeloperoxidase and proteinase 3 are the two major antigens present.
Tan and Peebles (37) in the Manual of Clinical Immunology describe a hemagglutination technique to quantitate antibodies to Sm and RNP. Durata and Tan (4), using saline-soluble extracts (ENA) from rabbit thymus acetone powder at a concentration of 5 mg protein/mL, demonstrated that increased sensitivity for detecting precipitating antibodies to RNP, Sm, and SS-B could be obtained by using CIE. A modified Ouchterlony technique has been used to show precipitating antibodies to RNA (35). Immunovision, Inc. has modified and tested the standard procedure (14) for enzyme immunoassays for the detection of autoantibodies using purified antigens.
There are many applications in the field of immunological monitoring in which the presence of body fluid antibodies and antigens are detected by a variety of methods. However, these assays usually measure one antibody or antigen at a time and tend to be time consuming and costly. Latex particles are commonly used clinically for detecting antibodies with agglutination as the end point. U.S. Pat. No. 5,162,863 discloses a method using flow cytometry to detect multiple antigens or antibodies with agglutination of particles combined with light scatter as the end point.
Microsphere based assays using flow cytometry have been reported by several investigators (20, 8) after Horan et al. reported the use of polystyrene microspheres to detect serum rheumatoid factor in 1979 (13).
The merger of bead assays with flow cytometry has been demonstrated in several clinical applications, e.g. detection of antibodies to CMV and herpes simplex (19); detection of antibodies to different components of the human immunodeficiency virus (HIV) (29); detection of antibodies to several antigens of Candida albicans (23); detection of human anti-mouse antibody (HAMA) in transplant patients receiving OKT3 (16); detection of circulating immune complexes (22) and HIV antibody in immune complexes (21); and detection of two different antibodies to CEA (17).
Although interest has focused on the detection of antibodies and antigens in fluids, the use of other ligand systems and biological probes has been explored, e.g. competitive binding of antibiotics to DNA coated beads (28) and detection of viruses (40).
Although the principals and advantages of fluorescent microsphere immunoassays have been discussed in the literature, applications in clinical lab testing have been relatively few despite the economics of time and cost inherent in this technology.
Current assays for the auto-antibodies seen in several autoimmune disorders are performed individually and require a separate kit for each antibody A method that will simultaneously assay for several different antibodies in one tube would be of significant value.
Hence, there is a need for an improved immunoassay method and apparatus for detecting and quantifying autoantibodies to nuclear antigens associated with autoimmune diseases as well as for detecting other antigens, antibodies, cell fragments, and the like.